This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Objectives: define (1) M-PMV Gag interactions with the cytoskeletal motor machinery, (2) role of viral and cellular components in transporting capsids from their assembly site to the plasma membrane, (3) capsid interactions with the cellular ESCRT machinery, and (4) the myristyl switch mechanism involved in budding. Efficient transport of pre-assembled capsids to the cell membrane requires a functional vesicular trafficking pathway and envelope (Env) glycoprotein. Transport may occur along a tubular-vesicular pathway induced by Env or another viral component, or these interactions may initiate Gag transport along other trafficking networks. We show Gag interacts in a CTRS-dependent manner with cellular Tctex-1, a light chain of the dynein motor complex, suggesting a microtubule-dependent retrograde transport to the pericentriolar region for capsid assembly. Microtubules might also explain anterograde transport of pre-assembled capsids to the plasma membrane. In M-PMV-infected CMMT cells, pulse-chase assays revealed a nocodazole induced 1-hour delay in virus production compared to untreated controls. Actin disruption led to a minor delay in virion release, while IF disruption had negligible effects. Live cell imaging showed that following microtubule disruption, an initial reduction in linear movement of capsids containing GFP-tagged Gag occurred, followed by a recovery of velocities. Nocodazole treatment led to a cessation in Env transport and deficient Env incorporation in released virions. Importantly, a continued albeit delayed release of virions was observed from cells lacking all three functional cytoskeleton components, suggesting a cytoskeleton-independent mode of transport. Studies are underway to alternate transport pathways. These studies could have implications for HIV/AIDS research.